Sofia Hassiotis

Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra‐cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally‐occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra‐cerebrospinal fluid sulphamidase delivery (100 μg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate‐oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra‐cerebrospinal fluid enzyme. Reductions in micro‐ and astrogliosis and delayed development of ubiquitin‐positive lesions were seen in both groups. A third group of intravenous‐only treated mice did not exhibit clinical or neuropathological improvements. Intra‐cerebrospinal fluid injection of sulphamidase effectively, but dose‐dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

Enzyme replacement reduces neuropathology in MPS IIIA dogs

There is no treatment for the progressive neurodegenerative lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA), which occurs due to a deficiency of functional N-sulfoglucosamine sulfohydrolase (SGSH), with subsequent accumulation of partially-degraded heparan sulfate and secondarily-stored compounds including GM2 and GM3 gangliosides and unesterified cholesterol. The brain is a major site of pathology and affected children exhibit progressive cognitive decline and early death. In the present study, six MPS IIIA dogs received intravenous recombinant human SGSH (rhSGSH) from birth to either 8 or 12 weeks of age (1 mg/kg, up to 5 mg), with subsequent intra-cerebrospinal fluid injection of 3 or 15 mg rhSGSH (or vehicle) on a weekly or fortnightly basis to 23 weeks of age. All dogs completed the protocol without incident, and there was no clinically-relevant cellular or humoral immune response to rhSGSH delivery. Immunohistochemistry demonstrated rhSGSH delivery to widespread regions of the brain, and tandem mass spectrometry revealed an apparent dose-dependent decrease in the relative level of a heparan sulfate-derived disaccharide, with near normalization of substrate in many brain regions at the higher dose. Secondarily-stored GM3 ganglioside and unesterified cholesterol, determined using histological methods, were also reduced in a dose-dependent manner, as was the number of activated microglia. We have demonstrated that pre-symptomatic treatment of this progressive neurodegenerative disorder via intra-cerebrospinal fluid injection of rhSGSH mediates highly significant reductions in neuropathology in this MPS IIIA model and clinical trials of this treatment approach in MPS IIIA patients are therefore indicated.

Impact of high-dose, chemically modified sulfamidase on pathology in a murine model of MPS IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder that results from a deficiency of sulfamidase (N-sulfoglucosamine sulfohydrolase), with consequential accumulation of its substrate, partially degraded heparan sulfate. Conventional doses (e.g. 1mg/kg) of intravenously delivered recombinant human sulfamidase (rhSGSH) do not improve neuropathology in MPS IIIA mice due to an inability to traverse the blood-brain barrier; however high-dose treatment or administration of enzyme that has been chemically modified to remove mannose-6-phosphate glycans has been shown to reduce neuropathology in related animal models. We have combined these approaches to evaluate the ability of 1, 5, 10 or 20mg/kg of similarly chemically modified or unmodified rhSGSH to reduce neuropathology following repeated intravenous delivery to adult MPS IIIA mice. rhSGSH was detected in brain homogenates from mice treated with all doses of modified rhSGSH and those receiving the two higher doses of unmodified rhSGSH, albeit at significantly lower levels. Immunohistochemically, rhSGSH visualized in the brain was localized to the endothelium, meninges and choroid plexus, with no convincing punctate intra-neuronal staining seen. This presumably underlies the failure of the treatment to reduce the relative level of a heparan sulfate-derived oligosaccharide (GlcNS-UA), or secondarily stored substrates that accumulate in MPS IIIA brain cells. However, modification of rhSGSH significantly increased its effectiveness in degrading GlcNS-UA in non-CNS tissues, potentially as a result of its reduced plasma clearance. If this observation is generally applicable, chemical modification may permit the use of significantly lower doses of lysosomal enzymes in patients currently receiving intravenous enzyme replacement therapy.

Allogeneic stem cell transplantation does not improve neurological deficits in mucopolysaccharidosis type IIIA mice

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative metabolic disorder caused by mutations in the N-sulfoglucosamine sulfohydrolase gene with resultant accumulation of partially degraded heparan sulfate (HS). Whilst allogeneic bone marrow transplantation (BMT) is indicated for several lysosomal storage disorders featuring neurodegeneration, its use in MPS III is highly controversial. Published evidence suggests that BMT does not improve cognitive function in MPS III patients. Despite this, patients continue to be transplanted in some centers. We therefore sought to determine the clinical effectiveness of BMT in a murine model of MPS IIIA. Pre-symptomatic young adult mice pre-conditioned with total body irradiation generated complete and stable donor-type chimerism. Whilst HS-derived disaccharides were reduced by up to 27% in the brain parenchyma, this was insufficient to decrease secondary cholesterol and GM3 ganglioside storage or permit clinical improvement. These results suggest that BMT is ineffective in its unmodified form and should not be considered as a treatment for MPS IIIA children.

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.

Delivery of therapeutic protein for prevention of neurodegenerative changes: comparison of different CSF-delivery methods.

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

Determination of the role of injection site on the efficacy of intra-CSF enzyme replacement therapy in MPS IIIA mice.

MPS IIIA is an inherited neurodegenerative lysosomal storage disorder characterized by cognitive impairment, sleep-wake cycle disturbance, speech difficulties, eventual mental regression and early death. Neuropathological changes include accumulation of heparan sulfate and glycolipids, neuroinflammation and degeneration. Pre-clinical animal studies indicate that replacement of the deficient enzyme, sulfamidase, via intra-cerebrospinal fluid (CSF) injection is a clinically-relevant treatment approach, reducing neuropathological changes and improving symptoms. Given that there are several routes of administration of enzyme into the CSF (intrathecal lumbar, cisternal and ventricular), determining the effectiveness of each injection strategy is crucial in order to provide the best outcome for patients. We delivered recombinant human sulfamidase (rhSGSH) to a congenic mouse model of MPS IIIA via each of the three routes. Mice were euthanized 24h or one-week post-injection; the distribution of enzyme within the brain and spinal cord parenchyma was investigated, and the impact on primary substrate levels and other pathological lesions determined. Both ventricular and cisternal injection of rhSGSH enable enzyme delivery to brain and spinal cord regions, with the former mediating large, statistically significant decreases in substrate levels and reducing microglial activation. The single lumbar CSF infusion permitted more restricted enzyme delivery, with no reduction in substrate levels and little change in other disease-related lesions in brain tissue. While the ventricular route is the most invasive of the three methods, this strategy may enable the widest distribution of enzyme within the brain, and thus requires further exploration.

A Preclinical Study Evaluating AAVrh10-Based Gene Therapy for Sanfilippo Syndrome

Mucopolysaccharidosis type IIIA (MPS IIIA) is predominantly a disorder of the central nervous system, caused by a deficiency of sulfamidase (SGSH) with subsequent storage of heparan sulfate-derived oligosaccharides. No widely available therapy exists, and for this reason, a mouse model has been utilized to carry out a preclinical assessment of the benefit of intraparenchymal administration of a gene vector (AAVrh10-SGSH-IRES-SUMF1) into presymptomatic MPS IIIA mice. The outcome has been assessed with time, measuring primary and secondary storage material, neuroinflammation, and intracellular inclusions, all of which appear as the disease progresses. The vector resulted in predominantly ipsilateral distribution of SGSH, with substantially less detected in the contralateral hemisphere. Vector-derived SGSH enzyme improved heparan sulfate catabolism, reduced microglial activation, and, after a time delay, ameliorated GM3 ganglioside accumulation and halted ubiquitin-positive lesion formation in regions local to, or connected by projections to, the injection site. Improvements were not observed in regions of the brain distant from, or lacking connections with, the injection site. Intraparenchymal gene vector administration therefore has therapeutic potential provided that multiple brain regions are targeted with vector, in order to achieve widespread enzyme distribution and correction of disease pathology.

Neonatal Bone Marrow Transplantation in MPS IIIA Mice

Patients with some neurological lysosomal storage disorders (LSD) exhibit improved clinical signs following bone marrow transplantation (BMT). The failure of mucopolysaccharidosis (MPS) type IIIA patients and adult mice with the condition to respond to this treatment may relate to factors such as impaired migration of donor-derived cells into the brain, insufficient enzyme production and/or secretion by the donor-derived microglial cells, or the age at which treatment is initiated. To explore these possibilities, we treated neonatal MPS IIIA mice with whole unfractionated bone marrow and observed that nucleated blood cell reconstitution occurred to a similar degree in MPS IIIA mice receiving green fluorescent protein (GFP)-expressing normal (treatment group) or MPS IIIA-GFP marrow (control group) and normal mice receiving normal-GFP marrow (control group). Further, similar distribution patterns of GFP(+) normal or MPS IIIA donor-derived cells were observed throughout the MPS IIIA mouse brain. We demonstrate that N-sulfoglucosamine sulfohydrolase (SGSH), the enzyme deficient in MPS IIIA, is produced and secreted in a manner proportional to that of other lysosomal enzymes. However, despite this, overall brain SGSH activity was unchanged in MPS IIIA mice treated with normal marrow and the lysosomal storage burden in whole brain homogenates did not decrease, most likely due to donor-derived cells comprising <0.24% of total recipient brain cells in all groups. This suggests that the failure of MPS IIIA patients and mice to respond to BMT may occur as a result of insufficient donor-derived enzyme production and/or uptake by host brain cells.

Low-dose, continuous enzyme replacement therapy ameliorates brain pathology in the neurodegenerative lysosomal disorder mucopolysaccharidosis type IIIA.

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained‐release (osmotic pump‐delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six‐week‐old MPS IIIA and unaffected mice were implanted with subcutaneous mini‐osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part‐way through the experimental period. We observed near‐normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump‐like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain.